Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function.

The continuous conversion of soluble fibronectin into extracellular matrix fibrils occurs through a dynamic, cell-dependent process. As the extracellular matrix is assembled, changes in the conformation of matrix proteins may expose biologically active, matricryptic sites that alter cell behavior. In this study, an in vitro model of wound healing was used to determine the role of matrix fibronectin in airway epithelial cell motility. Our findings indicate that, under basal conditions, small airway epithelial cell (SAEC) migration requires active fibronectin matrix polymerization. Furthermore, SAEC migration is increased significantly by the interaction of cells with a recombinant construct containing fibronectin's matricryptic III-1 site. In contrast, addition of increasing amounts of fibronectin to SAECs significantly decreased the rate of cell migration. This fibronectin-induced inhibition of cell migration was overcome by blocking excess fibronectin matrix deposition. These data indicate that SAEC migration is regulated in a biphasic manner by the polymerization of fibronectin in the extracellular matrix and suggest a stimulatory role for fibronectin's matricryptic III-1 site in cell motility.

The interaction of cells with the extracellular matrix (ECM) form of fibronectin (FN) triggers changes in growth, migration, and cytoskeletal organization that differ from those generated by soluble FN. As cells deposit and remodel their FN matrix, the exposure of new epitopes may serve to initiate responses unique to matrix FN. To determine whether a matricryptic site within the III1 module of FN modulates cell growth or cytoskeletal organization, a recombinant FN with properties of matrix FN was constructed by directly linking the cryptic, heparin-binding COOH-terminal fragment of III1 (III1H) to the integrin-binding III8–10 modules (glutathione-S-transferase [GST]–III1H,8–10). GST–III1H,8–10 specifically stimulated increases in cell growth and contractility; integrin ligation alone was ineffective. A construct lacking the integrin-binding domain (GST–III1H,2–4) retained the ability to stimulate cell contraction, but was unable to stimulate cell growth. Both GST–III1H,2–4 and matrix FN colocalized with caveolin and fractionated with low-density membrane complexes by a mechanism that required heparan sulfate proteoglycans. Disruption of caveolae inhibited the FN- and III1H-mediated increases in cell contraction and growth. These data suggest that a portion of ECM FN partitions into lipid rafts and differentially regulates cytoskeletal organization and growth, in part, through the exposure of a neoepitope within the conformationally labile III1 module.

During exercise, local mechanisms in tissues cause arterioles to rapidly dilate to increase blood flow to tissues to meet the metabolic demands of contracting muscle. Despite decades of study, the mechanisms underlying this important aspect of blood flow control are still far from clear. We now report a novel mechanism wherein fibronectin fibrils in connective tissue matrices transduce signals from contracting skeletal muscle to local blood vessels to increase blood flow. Using intravital microscopy, we show that local vasodilation in response to skeletal muscle contraction is specifically inhibited by an antibody that recognizes the matricryptic site in the first type III repeat of fibronectin (FNIII-1). In the absence of skeletal muscle contraction, direct application of FNIII-1–containing fibronectin fragments to cremaster muscle arterioles in situ, triggered a rapid, specific, and reversible local dilation that was mediated by nitric oxide and required the cryptic, heparin-binding sequence of FNIII-1. Furthermore, application of function-blocking FNIII-1 peptides to cremaster muscle arterioles rapidly and specifically decreased their diameter, indicating that the matricryptic site of fibronectin also contributes to resting vascular tone. Alexa fluor 488–labeled fibronectin, administered intravenously, was rapidly assembled into elongated, branching fibrils in the extracellular matrix of intact cremaster muscle, demonstrating active polymerization of fibronectin in areas adjacent to blood vessels. Together, these data provide the first evidence that a matricryptic, heparin-binding site within fibronectin fibrils of adult connective tissue plays a dynamic role in regulating both vascular responses and vascular tone.

Fibronectin is an adhesive glycoprotein that is polymerized into extracellular matrices via a tightly regulated, cell-dependent process. Here, we demonstrate that fibronectin matrix polymerization induces the self-assembly of multicellular structures in vitro, termed tissue bodies. Fibronectin-null mouse embryonic fibroblasts adherent to compliant gels of polymerized type I collagen failed to spread or proliferate. In contrast, addition of fibronectin to collagen-adherent fibronectin-null mouse embryonic fibroblasts resulted in a dose-dependent increase in cell number, and induced the formation of three-dimensional (3D) multicellular structures that remained adherent and well-spread on the native collagen substrate. An extensive fibrillar fibronectin matrix formed throughout the microtissue. Blocking fibronectin matrix polymerization inhibited both cell proliferation and microtissue formation, demonstrating the importance of fibronectin fibrillogenesis in triggering cellular self-organization. Cell proliferation, tissue body formation, and tissue body shape were dependent on both fibronectin and collagen concentrations, suggesting that the relative proportion of collagen and fibronectin fibrils polymerized into the extracellular matrix influences the extent of cell proliferation and the final shape of microtissues. These data demonstrate a novel role for cell-mediated fibronectin fibrillogenesis in the formation and vertical assembly of microtissues, and provide a novel approach for engineering complex tissue architecture.

The extracellular matrix protein, fibronectin stimulates cells to self-assemble into three-dimensional multicellular structures by a mechanism that requires the cell-dependent conversion of soluble fibronectin molecules into insoluble fibrils. Fibronectin also binds to collagen type I and mediates the co-assembly of collagen fibrils into the extracellular matrix. Here, the role of collagen-fibronectin binding in fibronectin-induced cellular self-assembly was investigated using fibronectin-null fibroblasts in an in vitro model of tissue formation. High resolution, two-photon immunofluorescence microscopy was combined with second harmonic generation imaging to examine spatial and temporal relationships among fibronectin and collagen fibrils, actin organization, cell proliferation, and microtissue morphology. Time course studies coupled with simultaneous 4-channel multiphoton imaging identified regional differences in fibronectin fibril conformation, collagen fibril remodeling, actin organization, and cell proliferation during three-dimensional cellular self-assembly. Regional differences in cell proliferation and fibronectin structure were dependent on both soluble fibronectin concentration and fibronectin-collagen interactions. Fibronectin-collagen binding was not necessary for either fibronectin matrix formation or intercellular cohesion. However, inhibiting fibronectin binding to collagen reduced collagen fibril remodeling, decreased fibronectin fibril extension, blocked fibronectin-induced cell proliferation, and altered microtissue morphology. Furthermore, continual fibronectin-collagen binding was necessary to maintain both cell proliferation and microtissue morphology. Collectively, these data suggest that the complex changes in extracellular matrix and cytoskeletal remodeling that mediate tissue assembly are driven, in part, by regional variations in cell-mediated fibronectin-collagen co-assembly.